63 research outputs found
Molecular characterization and immunomodulatory properties of MM cell-derived exosomes on NK cell-mediated functions: role of Toll-like receptor 2
Get PDFIntroduzione
Le cellule Natural Killer (NK) rappresentano una sottopopolazione di linfociti dell’immunita’ innata che svolgono un ruolo importante nel processo di immunosorveglianza dei tumori incluso il mieloma multiplo (MM), una neoplasia sostenuta dall’espansione clonale di plasmacellule maligne nel midollo osseo. Le cellule tumorali possono modulare le funzioni del sistema immunitario attraverso la secrezione sia di fattori solubili che di vescicole come gli esosomi. Tali microvescicole, formate nel compartimento endosomiale rappresentano un importante meccanismo di comunicazione tra cellule data la loro capacità di trasportare proteine, lipidi ed acidi nucleici.
Scopo del progetto
Scopo del mio progetto di dottorato e’stato quello di isolare e caratterizzare gli esosomi prodotti da cellule di MM sia in condizioni basali che in seguito a trattamento con il Melphalan, un agente genotossico utilizzato nella terapia del MM e di studiarne l’effetto sulle funzioni mediate dalle cellule NK.
Risultati
Il trattamento con il Melphalan determina un significativo aumento del rilascio di esosomi da parte di cellule di MM. La caratterizzazione srutturale e molecolare degli esosomi, effettuata utilizzando la microscopia elettronica, il western blot, e l’immunofluorescenza ed analisi al citofluorimetro mostra che queste vescicole esprimono molecole di derivazione endosomiale tra cui il CD63, il Tsg101, le molecole MHC di classe I, l’Hsp70 ed e’ importante notare l’assenza di calreticulina, una proteina esclusivamente presente nel reticolo endoplasmatico. Mediante analisi del profilo di espressione dei microRNA contenuti negli esosomi, ne abbiamo individuati alcuni potenzialmente in grado di modulare le funzioni delle cellule NK. Avvalendoci dell’analisi al microscopio confocale e al citofluorimetro, abbiamo osservato che gli esosomi sono captati e internalizzati da cellule NK e sono in grado d’indurre l’espressione del marcatore di attivazione CD69, di stimolare la produzione di INF-γ e di potenziare la proliferazione delle cellule NK indotta dall’IL-15. Il pre-trattamento delle cellule NK con l’SN50, un peptide che impedisce la traslocazione di NF-kB nel nucleo, determina l’inibizione della produzione di INF-γ indotta dagli esosomi suggerendo un coinvolgimento del “pathway” di NF-kB. Per individuare il meccanismo mediante cui gli esosomi attivano le cellule NK, ci siamo avvalsi dell’uso di cellule transfettate stabilmente con diversi recettori della famiglia toll (TLR) insieme al reporter luciferasi sotto il controllo di NF-kB. I nostri risultati dimostrano che gli esosomi inducono selettivamente l’attivita’ della luciferasi in cellule reporter esprimenti il recettore TLR2.
Conclusioni
I nostri risultati indicano che l’agente chemioterapico melphalan aumenta il rilascio degli esosomi da cellule di MM e che queste microvescicole hanno un ruolo immunostimulatorio sulle cellule NK. Abbiamo infine identificato il TLR2 come possibile “sensore” esosomiale. Questi risultati potrebbero essere importanti per migliorare l’efficacia dei protocolli di immunochemioterapia utilizzando gli esosomi per il potenziamento della risposta immunitaria innata
Efficacy of Milbemax (milbemycin oxime + praziquantel) in the treatment of dogs experimentally infected with Crenosoma vulpis
No full textCrenosoma vulpis, the fox lungworm, infects wild and domestic canids and is a cause of chronic respiratory disease in dogs in North America and Europe. The objective of this study was to determine the efficacy of milbemycin oxime (0.5mg/kg)/praziquantel (5mg/kg) (Milbemax; Novartis Animal Health, Inc.) against C. vulpis infection in a randomized, blinded, placebo-controlled study using experimentally infected dogs. Sixteen beagles (8 males, 8 females) were each given 100 infective third-stage larvae of C. vulpis. Fecal samples were examined for first-stage larvae by quantitative Baermann examination pre-exposure and at days 21, 28, 35, 42 and 49 post-infection (PI). All of the dogs were shedding larvae in the feces at 21 days PI. The dogs were randomly assigned to one of two groups. At 28 days PI, Group 1 (4 males, 4 females) received placebo only while Group 2 (4 males, 4 females) received a single treatment of milbemycin oxime (0.5mg/kg) and praziquantel (5mg/kg). The 16 dogs were euthanized and necropsied at 49 days PI. Lungs were removed, assessed for gross lesions (graded on a subjective scale 0-3 with 0 being normal) and C. vulpis were collected by lung-flush and counted. Samples of lung tissue were preserved for evaluation of histopathology and the lesions graded on a subjective scale (0-3 with 0 being normal). Gross and histopathology lesions were detected in all 8 untreated Group 1 dogs with mean subjective lesion scores of 1.8 ± 0.7 (range 1-3) and 3.0 ± 0.0 (range 3), respectively. Gross lesions were observed in 3/8 and histopathology lesions in all 8 of the treated Group 2 dogs with mean subjective lesion scores of 0.4 ± 0.5 (range 0-1) and 1.3 ± 0.4 (range 1-2), respectively. The mean (geometric) number for adult C. vulpis recovered in untreated dogs was 48.3 (range 25-70) compared with 0.65 (range 0-2) in animals treated with Milbemax. The resulting efficacy against C. vulpis was 98.7%. The number of C. vulpis was significantly lower for treated dogs than the burden in the untreated group (p=0.0002). A single dose of Milbemax (milbemycin oxime 0.5mg/kg+praziquantel 5mg/kg) was highly effective for the treatment of patent C. vulpis infection in dogs. A dosing interval for the prevention of clinical disease in dogs exposed to natural infections has not been established.journal articleresearch support, non-u.s. gov't2013 Dec 62013 09 20importe
Cancer extracellular vesicles as novel regulators of NK cell response
Get PDFNatural killer (NK) cells are innate lymphoid cells that play a major role in the immune surveillance against tumors and their activity is regulated through signals derived by a number of NK cell inhibitory and activating receptors as well as cytokines and other soluble factors released in the tumor microenvironment. Extracellular vesicles (EVs) are membrane-enclosed particles secreted by all cell types, both in healthy and diseased conditions, and are important mediators of intercellular communication. Depending on the molecular cargo, tumor-derived extracellular vesicles have the capability to either promote or suppress NK cell-mediated functions. Anti-cancer therapies designed to sustain host anti-tumor immune response represent an appealing strategy to control tumor growth avoiding tumor immune escape. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Herein, we will address the main mechanisms used by cancer-derived extracellular vesicles to modulate NK cell activity, and we will discuss how anti-cancer therapies might impact on the secretion and the immunomodulatory function of these vesicles
Cancer Exosomes as Conveyors of Stress-Induced Molecules: New Players in the Modulation of NK Cell Response.
Get PDFNatural killer (NK) cells are innate lymphoid cells that play a pivotal role in tumor surveillance. Exosomes are nanovesicles released into the extracellular environment via the endosomal vesicle pathway and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Emerging evidence has shown that stress stimuli provoke an increased release of exosome secretion. Remarkably, tumor-derived exosomes (Tex) produced in response to stress carry distinct type of DAMPs that activate innate immune cell populations. Moreover, stress-induced ligands for the activating receptor NKG2D are transported by this class of nanovesicles. Here, we will discuss how Tex interact with NK cells and provide insight into their potential role in response to chemotherapy-induced stress stimuli. The capability of some "danger signals" carried by exosomes that indirectly affect the NK cell activity in the tumor microenvironment will be also addressed
Distribution of Crenosoma vulpis and Eucoleus aerophilus in the lung of free-ranging red foxes (Vulpes vulpes)
No full textCrenosoma vulpis and Eucoleus aerophilus are nematode parasites that can cause verminous pneumonia in wild carnivores. There is a paucity of information regarding the distribution of parasites in the lungs and the relationship between histopathological and parasitological diagnoses in naturally infected foxes. The objectives of this study were: first, to study the lobar and airway distribution of C. vulpis and E. aerophilus in wild red foxes and second, to investigate the relationship between fecal and histopathological diagnoses. Samples from 6 sites of the lung and fecal contents were obtained from 51 wild foxes in Prince Edward Island. By fecal examination, 78.4% of wild foxes tested positive for C. vulpis and 68.6% for E. aerophilus. In contrast, 66.6% and 49% of foxes had histopathological evidence of C. vulpis and E. aerophilus in the lungs, respectively. Anatomically, C. vulpis was observed in the small bronchi and bronchioles of all pulmonary lobes whereas E. aerophilus was restricted to the large bronchi and the caudal lobes. Affected airways exhibited severe epithelial glandular hyperplasia and bronchiolar mucous metaplasia. It was concluded that C. vulpis is widely distributed in airways of all pulmonary lobes, whereas E. aerophilus is mainly restricted to the bronchi of caudal lobes. Also, this study showed that histological examination of lung underestimates the infection with E. aerophilus.PUBM: Print; JID: 9011490; ppublishSource type: Electronic(1
Infectivity of gastropod-shed third-stage larvae of Angiostrongylus vasorum and Crenosoma vulpis to dogs
No full textBackground
Metastrongyloid parasites Angiostrongylus vasorum and Crenosoma vulpis infect wild and domestic canids and are important pathogens in dogs. Recent studies indicate that gastropod intermediate hosts infected with various metastrongyloids spontaneously shed infective third-stage larvae (L3) into the environment via feces and mucus under laboratory conditions. Shed L3 retain motility up to 120 days, but whether they retain infectivity was unknown.
Methods
To assess the infectivity of shed L3, the heart/lungs of six red foxes (Vulpes vulpes) were obtained from trappers in Newfoundland, Canada. Lungs were examined for first-stage larvae (L1) by the Baermann technique. A high number of viable A. vasorum L1 and a low number of C. vulpis L1 were recovered from one fox; these were used to infect naïve laboratory-raised Limax maximus. L3 recovered from slugs by artificial digestion were fed to two naïve purpose-bred research beagles (100 L3/dog). L1 shed by these two dogs was used to infect 546 L. maximus (2000–10,000 L1/slug). L3 shedding was induced by anesthetizing slugs in soda water and transferring them into warm (45 °C) tap water for at least 8 h. Shed L3 recovered from slugs were aliquoted on romaine lettuce in six-well tissue culture plates (80–500 L3/well) and stored at 16 °C/75% relative humidity. Four naïve research beagles were then exposed to 100 L3/dog from larvae stored for 0, 2, 4, or 8 weeks, respectively, after shedding.
Results
All four dogs began shedding C. vulpis L1 by 26–36 days post-infection (PI). All four dogs began shedding A. vasorum L1 by 50 days PI.
Conclusions
L3 infectivity for the definitive host was retained in both metastrongyloids, indicating the potential for natural infection in dogs through exposure from environmental contamination. As an additional exposure route, eating or licking plant or other material(s) contaminated with metastrongyloid L3 could dramatically increase the number of dogs at risk of infection from these parasites.University of Prince Edward IslandElanco (United States
An in vitro larval motility assay characterizes anthelmintic efficacy against Crenosoma vulpis, Angiostrongylus vasorum, and Aelurostrongylus abstrusus
No full textOBJECTIVE
This study determined the in vitro efficacy of 6 common anthelmintics (eprinomectin, ivermectin, milbemycin oxime, moxidectin, selamectin, and fenbendazole) on motility (viability) of infectious third-stage larvae (L3) of Crenosoma vulpis, Angiostrongylus vasorum, and Aelurostrongylus abstrusus, which are important causes of canine and feline cardiopulmonary disease.
SAMPLES
First-stage larvae (L1) from C vulpis, An vasorum, and Ae abstrusus.
PROCEDURES
Naïve Limax maximus slugs were fed 1,000 to 2,000 L1 and held at 16 °C for at least 4 weeks to produce live L3. Approximately 50 to 100 L3/well were subsequently incubated in culture media alone or media containing 6 separate test anthelmintics at 4 concentrations, to bracket expected in vivo drug plasma levels in anthelmintic-treated dogs and cats. Drug effects on L3 motility (viability) were analyzed by multilevel logistic models, generating dose-response relationships. Experiments were completed 1-9/2019.
RESULTS
Drug concentration estimates corresponding to a 50% larval mortality rate identified that C vulpis was the most sensitive species to the anthelmintics tested. Ae abstrusus was most susceptible to moxidectin and selamectin, while An vasorum was insusceptible to all anthelmintics tested, except for selamectin at high drug concentrations.
CLINICAL RELEVANCE
The in vitro anthelmintic response to antiparasitic agents may guide and improve disease therapy and prevention. Considering the observed lack of efficacy against L3, monthly anthelmintic treatment for protection against An vasorum infection in dogs would primarily rely on the anthelmintic’s adulticidal activity. Maximal preventive control for An vasorum would, therefore, require at least 1 treatment administered a minimum of 1 week after the end of the transmission season
Crenosoma vulpis and the domestic dog: A study of prevalence on Prince Edward Island and of new diagnostic approaches
No full textCrenosoma vulpis is a metastrongyloid lung parasite which infects the bronchioles, bronchi, and trachea of wild and domestic canids and other carnivores. The infection cannot be diagnosed with standard fecal flotation examinations carried out at most veterinary clinics. Clinical signs of C. vulpis infection include chronic cough, wheezing, and decreased quality of life and closely mimic signs of canine allergic respiratory disease, which appears to be relatively common in dogs in the Atlantic Canadian region. Suspicions that the infection may have been previously misdiagnosed led to a study to determine the impact of C. vulpis infection on dogs on Prince Edward Island. The first objective was to determine the estimated prevalence of C. vulpis infection in PEI dogs. The second objective was to determine the proportion of PEI dogs infected with C. vulpis among those with clinical signs such as chronic cough. The third objective of the study consisted of developing a more sensitive diagnostic test, an enzyme-linked immunosorbent assay (ELISA), for the detection of antibodies to C. vulpis antigens in serum. (Abstract shortened by UMI.).Source: Masters Abstracts International, Volume: 37-03, page: 0883.Adviser: Gary Conboy
Targeting NKG2D and NKp30 ligands shedding to improve NK cell–based immunotherapy
Get PDFNatural killer (NK) cells are critical immune effector cells capable of mediating antitumor responses. These cytotoxic lymphocytes recognize transformed cells through a mechanism mainly dependent on the engagement of several activating receptors. However, many tumors have developed strategies to evade immunosurveillance and detection by NK cells. A relevant immune escape mechanism is the down regulation of NK cell activating ligands on the surface of tumor cells by proteolytic shedding mediated by different members of metalloproteinase families. Here, we consider two important NK activating receptors, namely NKG2D and NKp30, the ligands (i.e., MICA/B, ULBPs, and B7-H6) of which can be released by cancer cells through proteolytic cleavage. Modulation of ligand shedding in response to cancer therapy is also examined, and we discuss how metalloproteinases implicated in the ligand cleavage could be targeted in novel therapeutic schemes to counteract tumor escape from stress-elicited immune responses
Increased oxydative stress contribute to cardiomyocyte dysfunction in patients with Fabry disease cardiomyopathy.
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